For convenience, publications relating to the following description of the background of the invention are numerically referenced and listed in the appended bibliography.
Evidence (see references 1 to 7) has previously been reported indicating that at least some malignant tumours in mammals, especially cachexia-inducing tumours, give rise to the production of one or more catabolic factors which may be found in the circulatory system, e.g. in blood plasma.
It is well known that cachexia, characterised by progressive weakness, dramatic weight loss and wasting, is a common condition arising in many human cancer patients, especially in patients with gastrointestinal or lung cancer, and this often appears to be the most frequent cause of eventual death in such patients. Since, however, cachexia at least in human patients generally arises, often at an early stage, when the tumour mass is only a very small proportion of body weight it cannot be generally explained by a simple competitive effect between the tumour and body tissues for available nutrients; also, although in some cases cancer cachexia is accompanied by anorexia manifested by a severely reduced food and water intake, anorexia does not occur in all cases or appears only after severe loss of weight and body tissues (fat and muscle) has already become established; anorexia cannot therefore be recognised as a primary cause of all or many cancer cachectic conditions, and it seems possible that catabolic factors arising from the tumour and acting more directly on the host tissues may be primarily responsible.
Additionally, there is also some evidence indicating that growing tumours may derive at least part of the fatty acids they require, e.g. for membrane formation, from tissues of their host, and that in at least some cases these fatty acids may be derived from the fats in adipose tissue of the host through the action of a lipolytic catabolic factor that may be found in the host's circulatory system in the presence of the tumour. However, although this may be deemed suggestive of a possible link or causal relationship between such lipolytic factor, other catabolic factors possibly present, the growing tumour, and symptoms of cachexia independent of anorexia, various previous attempts (references 3, 4, 5, 6 and 7) to isolate, identify and characterise a lipolytic factor of this kind have produced somewhat confusing, uncertain, and inconsistent results and there has also been substantial doubt as to the extent of dependence of any such lipolytic factor on tumour specificity.
Thus, in 1980 it was reported by Kitada et al (reference 3) that the serum of AKR mice bearing thymic lymphoma contained a potent lipid mobilizing factor having lipolytic activity as evidenced by comparative in vivo assaying of the breakdown of adipose tissue labelled with radioactive carbon implanted into test animals of which some were injected with samples of the serum, measurements being taken of the radioactive carbon appearing in the respiratory CO.sub.2. The same effects of breakdown of the implanted adipose tissue were also observed upon injecting samples of extracts of the tumours and also upon injecting samples of culture medium from an AKR mouse lymphoma cell culture. These results were also reported again in 1981 (reference 4) by the same investigators, together with the additional result of a similar adipose tissue breakdown effect being observed upon in vivo assaying in the same way a serum sample from a human cancer patient with adenocarcinoma. In addition, the results then reported (somewhat superfically) also included the results of a preliminary attempt to isolate and characterise the active substance by a gel filtration technique involving chromatographing a dilute acetic acid extract of the thymic lymphoma tumour tissue using a Bio-Gel P6 column from which the various fractions were again tested for lipolytic activity by the same in vivo assay technique. It was then deduced from these results that the active lipolytic factor was a small "heat stable" protein having a molecular weight of about 5000 daltons. However, this conclusion was not substantiated by later results (see below) and, in any event, it will be appreciated that use of the in vivo assaying technique did not necessarily exclude the possibility that the test samples injected merely triggered the production of, or activated, a lipolytic factor within the test animal instead of the test samples themselves containing the active lipolytic factor.
Subsequently, Kitada et al reported in 1982 (reference 5) that after continuing their investigations using extracts of thymic lymphoma tumour tissue from AKR mice, using an in vitro technique for assaying lipolytic activity involving the measurement of liberated glycerol after incubation of samples of the tumour tissue extracts with preparations of rat adipocytes, lipolytic activity could only be detected after ageing of the extracts kept at low temperature (4.degree. C.) for several days. Following chromatographic gel filtration of such aged active extracts, whose activity was found to be completely destroyed by digesting with trypsin, they then concluded that the lipolytic active substance which they had detected was formed by aggregation of inactive small protein molecules.
The presence of a lipolytic factor in ascites fluid from DDK mice with sarcoma 180 and in ascites fluid from human cancer patients with hepatoma has also been reported by Masuno et al (references 6 and 7) but in this case their experimental evidence indicated that the lipolytic factor found, termed Toxohormone-L, was an acidic protein of high molecular weight (of the order of 65,000 to 75,000 daltons) which acted indirectly by suppressing food and water intake, thereby promoting anorexia as the main cause of breakdown of adipose tissue and symptoms of cachexia.
Similarly, a macrophage product tumour necrosis factor (TNF) and the homologous or related substance cachectin (see references 10 and 11) which inter alia inhibit lipoprotein lipase activity and induce weight loss have also been implicated as agents concerned in causing cancer cachexia, but again any cachectic effects arising from this source appear to be caused primarily by anorexic effects or dehydration (see reference 12).
It is necessary to recognise, however, that the conditions in experimental animals such as mice and rats bearing tumours of the kind mentioned above may not properly reflect the conditions present in human cancer patients afflicted with cachexia-inducing tumours, especially bearing in mind that in rodents many such tumours grow relatively rapidly and that evidence of cachexia is often apparent, if at all, only at a stage when the tumour has reached a size equivalent to 30-40% of total body weight. In contrast, in humans tumour growth is slower and tumour mass rarely reaches or exceeds 5% of total body weight although symptoms of cachexia often arise whilst the tumour mass is but a small fraction of 1% of total body weight. Nevertheless, there has been a promising development for improving the conditions for experimental investigations following studies more recently reported (references 1 and 2) on mice (pure NMRI strain) bearing a tumour designated MAC16, first described by Cowen et al (see reference 8), of an established series (MAC) of chemically induced, transplantable colon adenocarcinomas, this MAC16 tumour being produced by a particular cell line now deposited on Mar. 8, 1989 in the European Collection of Animal Cell Cultures (ECACC) at the Public Health Laboratory Service Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, United Kingdom under a provisional accession number 8903016.
The MAC16 tumour is a moderately well-differentiated adenocarcinoma which has been serially passaged in mice for many years, and it has been found that it appears to represent a more satisfactory experimental model for tumours which induce cachexia in human patients, especially insofar as it has often been found to produce substantial loss of body weight at small tumour burdens (less than 1% body weight) and without a reduction in the intake of either food or water (reference 1).
Some of these recently reported studies (reference 2) have further indicated that the MAC16 tumour in mice gives rise to the production of circulatory catabolic factors, apparently comprising both a lipolytic factor and a proteolytic factor, which seem to be present in the plasma of the tumour-bearing animals and which, it could be postulated, might be directly responsible at least to some extent for the breakdown of the body fat and muscle tissues, and hence for symptoms of cachexia. More particularly, the lipolytic factor was reported as having a lipolytic activity (as measured either in tumour extracts or plasma samples incubated with mouse epididymal adipose tissue followed by use of an in vitro free fatty acid assaying technique) which is non-dialysable, which is destroyed by heat and acid, and which is inhibited by insulin and 3-hydroxybutyrate. However, no more precise characterisation or isolation of this factor was reported, although in a later paper (reference 12) evidence has been presented showing that it is not the same as, and is clearly distinguished from, cachectin or tumour necrosis factor (TNF) referred to earlier.